Framework for a Physical Map of the Human 22q13 Region Using Bacterial Artificial Chromosomes (BACs)

GENOMICS 33, 9–20 (1996)ARTICLE NO. 0154

Framework for a Physical Map of the Human 22q13 Region UsingBacterial Artificial Chromosomes (BACs)

HOLGER SCHMITT,*,† UNG-JIN KIM,* TATIANA SLEPAK,* NIKOLAUS BLIN,†MELVIN I. SIMON,* AND HIROAKI SHIZUYA*,1

*California Institute of Technology, Division of Biology 147-75, Pasadena, California 91125; and †Institut fur Anthropologie undHumangenetik, Universitat Tuebingen, Wilhelmstrasse 27, 72074 Tuebingen, Germany

Received November 7, 1995; accepted January 19, 1996

Despite their important role in the construction ofDetailed physical maps of entire chromosomes based physical maps of entire chromosomes (Chumakov et al.,

on combined genetic, cytogenetic, and structural in- 1992; Foote et al., 1992; Nizetic et al., 1994; Bell et al.,formation are essential components for positional 1995), a high rate of chimerism and clonal instabilitycloning and genomic sequencing. Despite the wealth often restrict the reliability of YACs for mapping andof genetic information of the known diseases in the sequencing purposes (Green et al., 1991; Kouprina etchromosome 22q13, the construction of a detailed al., 1994; Larionov et al., 1994; Neuhausen et al., 1994).physical map of the terminal region is difficult due to On the other hand, established bacterial vector–hostthe sparsity of the genetic markers. We present here systems like cosmids (de Jong et al., 1989), Fosmidsa map of bacterial artificial chromosome (BAC) contigs (Kim et al., 1992), and P1 (Sternberg, 1990; Pierce etthat cover a number of genetic loci in the 22q13 region. al., 1992) allow easier access to detailed molecularOne hundred thirty-six BACs with an average insert

analysis of the cloned fragments but can propagate onlysize of 140 kb are assembled into 35 contigs defined byrelatively small inserts (40–100 kb), thus making the64 markers in 22q13–qter. Twenty-three anonymousconstruction of extended contigs a laborious effort.markers are now linked to the previously mapped ge-

As an alternative to these systems, we developed anetic anchor points. q 1996 Academic Press, Inc.bacterial cloning system based on the Escherichia coliF-factor replicon called the bacterial artificial chromo-some (BAC)2 system (Shizuya et al., 1992). The BACINTRODUCTIONsystem provides the stable propagation and mainte-nance of relatively large (ú300 kb) genomic DNA frag-The construction of integrated physical and geneticments as single-copy plasmids in the well-character-linkage maps is of basic interest in the ongoing effortized recombination-deficient host strains. High stabil-to map and sequence the entire human genome. Contig-ity, minimal chimerism, and ease of purification ofuously overlapping sets of genomic clones spanninglarge inserts characterize the BAC vector system as along regions of the genome that are defined by anchorsuitable source of intact DNA fragments for con-points on a genetic linkage map will substantially facil-structing large-scale detailed physical maps of genomicitate the localization and identification of new diseaseregions. We have constructed a total human BAC li-genes and thus allow focused studies of gene expressionbrary that to date is represented by 235,000 individualon a molecular level. High-density physical maps relateclones with an average insert size of nearly 140 kbthe information from genetic linkage analysis to de-providing approximately 101 coverage of the entire hu-tailed molecular characterization of genes, transcripts,man genome (Shizuya et al., manuscript in prepara-and chromosome structure. Furthermore, they providetion). A portion of the library (160,000) has beengenomic DNA templates for sequencing individualprinted on nylon filters in high-density format to allowchromosomes.efficient screening by colony hybridization and has alsoLong-range physical mapping can be establishedbeen formatted in pools of clones for rapid PCR screen-most efficiently using cloning systems capable of car-ing. The library is currently being used in our lab torying very large fragments (ú1 million bp) of exoge-develop a contiguous physical map of the entire longnous DNA in a single clone. Yeast artificial chromo-arm of human chromosome 22.somes (YACs; Burke et al., 1987) represent such a sys-

tem but have the disadvantage of inconvenient2 Abbreviations used: ACO, Aconitase; ARSA, arylsulfatase A;manipulation and difficult isolation of the cloned DNA.

BAC, bacterial artificial chromosome; BZRP, peripheral benzodia-zapine receptor; CYP2D6, debrisoquine 4-hydroxylase (cytochromeP450IID6); DIA1, cytochrome b5 reductase (diaphorase); YAC, yeast1 To whom correspondence should be addressed. Telephone: (818)

395-4154. Fax: (818) 796-7066. artificial chromosome.

90888-7543/96 $18.00

Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

AID Genom 3994 / 6r12$$$221 02-29-96 00:53:06 gnmal AP: Genomics

SCHMITT ET AL.10

TABLE 1

22q13 Probes Used to Screen the BAC Library by Hybridization

Locus/probe name Insert (kb) Restriction site/vector Localization

ACO/I1 (Zheng et al., 1990) 0.9 EcoRI/pUC18 q11.2–q13.3ARSA/pCP8 (Stein et al., 1989) 1.4 EcoRI/pBluescript q13.31–qterBZRP/p-hPBS11 (Riond et al., 1991) 0.84 BamHI-HindIII/pTZ19 q13.31–qterCYP2D6/p91023(B) (Gonzales et al., 1988,

Gough et al., 1993) 1.8 EcoRI/pUC19 q13.1DIA1/pb5R141 (Yubisui et al., 1987) 1.8 EcoRI/pUC13 q13.1–qterD22S16/p22hom13 (Goettert et al., 1989) 1.2 BamHI–EcoRI fragment/pTZ18 q13.1D22S21/W13E (Rouleau et al., 1989) 3.6 HindIII/pUC19 q13D22S22/W110D (Rouleau et al., 1989) 1.9 HindIII/pUC19 q13D22S23/W24F (Rouleau et al., 1989) 2.1 HindIII/pUC19 q13D22S34/pH 7 (Budarf et al., 1991) 1.2 HindIII/pUC18 q11.2–qterD22S35/pH 10 (Budarf et al., 1991) 1.8 HindIII/pUC18 q11.2–qterD22S40/pH 19 (Budarf et al., 1991) 5.0 HindIII/pUC18 q11.2–qterD22S45/pH 41a (Budarf et al., 1991) 1.4 HindIII/pUC18 q11.2–qterD22S55/pH 91 (Budarf et al., 1991) 5.5 HindIII/pUC18 q13–qterD22S64/pH 130 (Budarf et al., 1991) 2.8 HindIII/pUC18 q11.2–qterD22S82/KI-63 (Dumanski et al., 1990) 2.1 HindIII/Charon 21A q13–qterD22S84/KI-216 (Dumanski et al., 1990) 3.0 HindIII/Charon 21A q13–qterD22S87/KI-120 (Dumanski et al., 1990) 0.73 HindIII/Charon 21A q12–q13D22S91/KI-211 (Dumanski et al., 1990) 6.7 HindIII/Charon 21A q12–q13D22S92/KI-218 (Dumanski et al., 1990) 1.1 HindIII/Charon 21A q12–q13D22S94/KI-1105 (Dumanski et al., 1990) 1.95 HindIII/Charon 21A q13–qterD22S95/KI-839 (Dumanski et al., 1990) 2.0 HindIII/Charon 21A q13–qterD22S98/K1-1149 (Dumanski et al., 1990) 2.1 HindIII/Charon 21A q12–q13D22S102/KI-436 (Dumanski et al, 1990) 2.1 HindIII/Charon 21A q12–q13D22S106/KI-1543 (Dumanski et al., 1990) 0.6 HindIII/Charon 21A q12–q13D22S157/KI-536 (Dumanski et al., 1990) 2.2 HindIII/Charon 21A q13–qterD22S163/MS607 (Armour et al., 1990) 2.3 EcoRI fragment q13–qterD22S170a 0.55 EcoRI–HindIII fragment/pTZ18 q12–q13D22S326/DAC9 (Lamour et al., 1993) 1.3 XbaI–BamHI fragment/Lawrist q13–qterD22S453-D22Z6/N62A6 (Xie et al., 1994) Ç40 EcoRI/Lawrist q13.3–qterD22S455/N67E5 (Xie et al., 1994) Ç40 EcoRI/Lawrist q13.3–qterKI-44b 0.61 HindIII/Charon 21A q12–q13KI-60b 1.8 HindIII/Charon 21A q13–qterKI-61b 3.5 HindIII/Charon 21A q13–qterKI-175b 2.8 HindIII/Charon 21A q13–qterKI-385b 2.3 HindIII/Charon 21A q13–qterKI-487b 3.0 HindIII/Charon 21A q12–q13KI-775b 2.0 HindIII/Charon 21A q12–q13KI-800b 1.6 HindIII/Charon 21A q12–q13KI-830b 3.0 HindIII/Charon 21A q13–qterKI-859b 0.5 HindIII/Charon 21A q12–q13KI-874b 1.1 HindIII/Charon 21A q12–q13KI-932b 0.5 HindIII/Charon 21A q12–q13KI-1017b 1.85 HindIII/Charon 21A q12–q13KI-1210b 1.85 HindIII/Charon 21A q13–qterKI-1528b 0.98 HindIII/Charon 21A q13–qterKI-1586b 1.9 HindIII/Charon 21A q13–qterKI-1731b 2.2 HindIII/Charon 21A q12–q13

a G. Thomas, pers. comm.b J. P. Dumanski, pers. comm.

Several clinically relevant disorders and neoplasias syndrome (Driscoll et al., 1992), and the cat eye syn-drome (McDermid et al., 1986) are caused by deletionsare associated with anomalies involving chromosome

22. There are a number of important genes involved in or duplications of parts of 22q11. Deletions of variousextent were frequently observed in meningiomas, neu-a variety of tumor-related translocations, i.e., in

chronic myelogenous leukemia, acute lymphocytic leu- rofibromatosis type 2, and acoustic neuromas (Sei-zinger et al., 1986; Emanuel et al., 1993) suggestingkemia, Burkitt lymphoma, Ewing sarcoma (reviewed in

Kaplan et al., 1987), and soft tissue clear cell sarcoma the presence of tumor suppressor genes in 22q.Since most clinical disorders mentioned above in-(Delattre et al., 1992; Zucman et al., 1993). Several

developmental disorders such as the DiGeorge syn- volve chromosomal alterations in 22q11 and 22q12,these regions have been well studied at both the cytoge-drome and its more complex form of disorder called

CATCH22 (Wilson et al., 1993), the velocardiofacial netic and the molecular levels. The majority of the


22q13 PHYSICAL BAC MAP 11

FIG. 1. Autoradiogram of BAC library screening with 22q13 region-specific pools of probes by colony filter hybridization. Only 8 froma total of 42 filters of the human BAC library gridded into high-density format are shown.

Hudson and S. Foote (Whitehead Institute/MIT Center for Genomeknown chromosome 22-specific markers are assignedResearch). Primers for the following STSs were used for PCRto the more proximal part of the long arm, enablingscreening of library pools as described elsewhere (Shizuya et al.,the establishment of high-coverage physical maps manuscript in preparation): D22S546/WI-424; D22S559/WI-113;

(Scambler, 1994; Bell et al., 1995). However, due to low D22S578/WI-17; D22S611/WI-30; and D22S645/WI-382. Subse-marker density, the distal portion remains less charac- quent clones from a chromosome 22-specific Fosmid library (Kim et

al., 1992), localized by fluorescence in situ hybridization (FISH)terized, leaving the largest gap in 22q13 (Scambler,analysis (B. Birren et al., manuscript in preparation) were used as1994). Terminal deletions in several clinical caseshybridization probes: F1A6, F1A7, F1B11, F1E1, and F2C6(Watt et al., 1985; Herman et al., 1988; Kirschebaum (22q13.3); F1C11, F1F9, and F1G11 (22q13.1–q13.2); F1E3 (22q12–

et al., 1988; Romain et al., 1990; Narahara et al., 1992; q13.1); and F2G8 and F3D4 (22q).Phelan et al., 1992) and a loss of heterozygosity in a Probe preparation and BAC characterization. Plasmid and cos-22q13.3 locus in two neurofibromatosis patients (Tom- mid DNA was isolated from 5-ml cultures supplemented with themerup et al., 1992) were reported. In addition, a critical suitable antibiotic and purified by Qiagen columns (Qiagen, Inc.,

Chatsworth, CA). As the vectors of the plasmids and cosmids usedregion for the del(22)(q13.3) syndrome has been definedin this study do not share any sequences with the BAC vector, they(Nesslinger et al., 1994), and cases of mental retarda-did not have to be removed for hybridization. In cases where onlytion were linked to subtelomeric regions on chromo-fragments of the insert DNA were used as probes, plasmid or cosmid

some 22 (Flint et al., 1995). In this study, we present DNA was digested with the appropriate restriction enzyme (Newthe establishment of extended BAC contigs in chromo- England Biolabs), and bands were excised after agarose gel electro-

phoresis was performed. DNA was purified using a gel extraction kitsomal region 22q13, which provide the backbone for(Qiagen). Human DNA inserts from KI probes were amplified bycomplete physical coverage and detailed molecularPCR as described in Dumanski (1990). PCR products were run oncharacterization of this particular genomic region.low-melting-point agarose gels, and product bands were excised.Agarose plugs were then melted, and an aliquot was used for radioac-tive labeling. Fosmid and BAC DNA was prepared by a standardMATERIALS AND METHODSalkaline lysis procedure (Sambrook et al., 1989) with the automatedDNA isolation system AutoGen 740 (Integrated Separation Systems,Probes. The probes used to screen the human BAC library byNatik, MA). To use it as hybridization probe to screen the BAC li-colony hybridization are listed in Table 1. Single-copy DNA probesbrary, insert DNA was prepared to avoid vector cross-hybridization.(KI probes) from a chromosome 22-specific bacteriophage l genomicDNA preparations were digested to completion with NotI restrictionlibrary were obtained from J. P. Dumanski (Karolinska Institute).endonuclease (New England Biolabs), and fragments were separatedSequence information about STSs localized in chromosomal region

22q13 (WI STSs; Hudson et al., 1994) was kindly provided by T. by PFGE (CHEF-DR III; Bio-Rad, Hercules, CA) at a field strength


SCHMITT ET AL.12

TABLE 2a

BAC Contigs Identified by Mapped Genetic Markers

NotI InsertD No. or gene NotI fragments sizeprobe name BACs sites (kb) (kb) Other markers present

D22S102 256C5 1 160, 40 200 F1F9, F1G11KI-436 833B7 nd

D22S91 301B10 0 135 135 D22S92/KI-218, D22S578/WI-17, KI-800KI-211D22S92 301B10 0 135 135 D22S91/KI-211, D22S578/WI-17, KI-800KI-218 424D11 1 135, 20 155 D22S645/WI-382

424D12 1 130, 65 195 D22S578/WI-17, D22S645/WI-382, KI-800472H1 1 75, 70 145 D22S578/WI-17, D22S645/WI-382, KI-800509A8 1 110, 35 145 D22S578/WI-17, D22S645/WI-382, KI-800707D1 0 160 160 D22S578/WI-17, KI-800917G6 1 180, 10 190 D22S645/WI-382, KI-800

D22S645 29F11 1 150, 40 190WI-382 206C7 1 75, 30 105

D22S87 214G2 0 190 190 KI-44KI-120 571A10 0 110 110 KI-44

573A11 0 110 110 KI-44KI-44 374A3 1 200, 35 235

385D10 0 120 120386E12 0 120 120

571A10 159A9 0 170 170189B5 nd224H3 nd373G5 0 165 165

D22S95 567E5 0 120 120KI-839 888F8 1 120, 30 150 F1B11

D22S64 475G12 1 130, 10 140pH 130

CYP2D6 448G12 ndp91023(B) 521D12 0 60 60

570E4 0 90 90

D22S40 140G2 2 175, 55, 30 260 F1A7pH 19 354D11 nd

397C4 0 50 50

D22S22 104F1 1 150, 30 180 D22S35/pH 10, KI-1210, KI-1586W110D 157A5 1 82, 60 142 D22S35/pH 10, KI-1210, KI-1586, F1E1

377B11 3 70, 60, 33, 30 193 D22S35/pH 10, KI-1210, KI-1586426B6 1 130, 90 220 D22S35/pH 10, KI-1210, KI-1586, F1E11028C9 nd D22S35/pH 10, KI-1210, KI-1586

KI-1210 801E10 0 80 80426B6 24D6 0 50 50 D22S611/WI-30

436F11 0 140 140

D22S94 358H9 0 140 140KI-1105 744H10 0 90 90

899G1 0 165 165

D22S157 1054F7 1 120, 40 160KI-536

D22S82 89D12 ndKI-63 144G4 nd

D22S84 278B7 0 200 200 D22S546/WI-424, KI-61KI-216KI-61 164G5 nd

280A3 ndD22S45 299D3 2 65, 30, 20 115 D22S34/pH 7pH41a

AID Genom 3994 / 6r12$$3994 02-29-96 00:53:06 gnmal AP: Genomics


TABLE 2a—Continued


D22S23 115C9 2 180, 8, 2 190 D22S453-D22Z6/N62A6, D22S455/N67E5W24F115C9 239G3 2 120, 10, 8 138

988D11 2 75, 60, 10 145988D11 148G1 1 80, 40 120 F2G8, F3D4

406C6 0 50 50722E9 1 80, 50 130 F2G8, F3D4969G12 2 90, 40, 20 150

D22S21 84F1 0 90 90W13E 333D4 0 155 155

639A7 0 110 110

D22S55 34B1 1 130, 90 220 D22S326/DAC9pH 91 276D9 2 130, 75, 15 220 D22S326/DAC9, F1A6

698D7 0 180 180276D9 230C2 0 130 130

567H2 0 160 160B32H3 0 130 145

ARSA 384D8 3 40, 35, 30, 25 130pCP8384D8 88H6 3 40, 30, 20, 15 105

325F5 0 45 45999D10 2 40, 30, 15 85B129E11 1 45, 40 95

D22S163 799F10 2 60, 35, 30 125MS607

Note. Previously ordered genetic anchor points (Dumanski et al., 1991) are displayed in boldface characters and can be found togetherwith corresponding BAC contigs in Fig. 2. The probes are ordered from most proximal to most distal.

of 6 V/cm on 11 TAE–1% low-melting-point agarose gels (SeaPlaque, gested with HindIII and EcoRI and separated on 0.7% agarose gels.If necessary to confirm continuity, Southern hybridization of theFMC Bio Products, Rockland, ME) with a linear pulse from 5 to 15

s for 18 h at 147C. Insert fragment sizes were determined by direct probe or BAC DNA to blotted BAC digests was performed in 15%formamide, 0.2 M Na2HPO4, 1 mM EDTA, 1% BSA, and 7% SDScomparison with l concatemers (New England Biolabs). Insert bands

were excised, and agarose plugs were melted at 657C, cooled to 457C, overnight at 457C. Stringent washes were as described for colonyhybridization.and digested to completion with 1 U GELase (Epicentre Technolo-

gies, Madison, WI) per 100 ml melted agarose. DNA was ethanol-precipitated and resuspended in TE buffer. All probes were radiola-beled with [32P]dATP by random priming (Feinberg and Vogelstein, RESULTS AND DISCUSSION1983) and purified on Sephadex G-50 spin columns.

Total genomic BAC library screening. Library filters were prehy- Physical Mapping Using BAC Clonesbridized at 657C for 30 min in hybridization buffer containing 1 MNaCl, 50 mM Tris–HCl, pH 8.0, 5 mM EDTA, 1% SDS, and 10%

We used a complex set of chromosome 22 region-dextran sulfate. Probes likely to contain repetitive sequences werespecific DNA probes to screen the total BAC library topreannealed with sonicated human placental DNA (Sigma) in hy-

bridization buffer at 657C for 30 min. Colony hybridization was per- establish a collection of genomic BAC clones corre-formed for 16 h at 657C in the same buffer with a minimum of 104 sponding to previously identified markers. Screeningcpm/ml of 32P-labeled probe. Filters were washed twice each in 21 probes include genetic markers previously ordered bySSC, 0.1% SDS at 427C followed by 0.11 SSC, 0.1% SDS at 657C and

linkage analysis, unique genomic loci such as cDNAs,exposed to X-ray films (Kodak X-Omat AR) at 0707C for 1–3 days.and anonymous probes localized to subchromosomal re-Probes were stripped off in 1 mM EDTA, pH 8.0–0.1% SDS at 857C,

and the library filters were stored in 51 SSC at 47C until reuse. gions by somatic cell hybrid panels or FISH mapping.Analysis of positive BAC clones and contig formation. Positively A summary of the hybridization probes used to screen

identified BAC clones were picked from the library microtiter dishes, the library filters and their localization on chromosomestreaked out onto LB plates supplemented with 12.5 mg/ml chloram- 22 is shown in Table 1. In addition, PCR screeningphenicol, and grown overnight at 377C. Two different colonies from

of library pools with STS primers was performed (seeeach clone were used to inoculate 3 ml liquid medium, and culturesMaterials and Methods).were grown overnight. Prior to the addition of new BAC clones to

the chromosome 22-specific sublibrary, BAC DNA was isolated as A total of 64 different probes from chromosomal sub-described, and integrity was checked by HindIII digestion. Individual region 22q13 identified 136 BAC clones. Relying on re-hybridization was performed either on bacterial colonies gridded onto striction fingerprint analysis and Southern hybridiza-nylon filters (chromosome 22-specific BAC sublibrary) or by dot hy-

tion data, 35 contigs containing between 1 and 10 BACbridization on BAC DNA spotted onto nylon filters. For contig forma-tion, clones positive for one probe were collected, and DNA was di- clones were assembled. This preassignment of ordered


SCHMITT ET AL.14

TABLE 2b

BAC Contigs Identified by Other 22q13 Region-Specific Markers


ACO 179H8 0 75 75I1 242G8 0 175 175

243C12 1 185, 35 220260C11 0 220 220920C4 0 140 140960C9 0 140 1401155C2 0 180 180

BZRP 390D3 2 90, 30, 10 130p-hPBS11 821B11 3 75, 40, 35, 25 175 KI-1528KI-1528 1164F10 2 50, 45, 30 125821B11 633D5 0 50 50

1178H7 nd1191B2 2 90, 50, 30 170

DIA1 10B8 1 185, 15 200 D22S559/WI-113, F1C11pb5R14.1 10D8 1 185, 15 200 D22S559/WI-113, F1C11

488B11 3 80, 75, 35, 25 215F1C11 117H8 nd D22S559/WI-113D22S559 35C2 0 150 150WI-113

D22S16 585A10 2 60, 30, 28 118p22hom13 694C8 2 80, 50, 30 160

D22S106 264H6 0 55 55KI-1543 443B1 0 230 230443B1 212A2 1 220, 25 245 D22S98/KI-1149D22S98 23B3 0 50 50KI-1149

D22S170 113D3 nd113D4 0 160 160113G8 0 160 160

113D4 150C2 0 200 200374E12 0 145 145

KI-60 204B7 0 85 85617H6 0 160 160

KI-175 150B4 0 160 160 F2C6343C1 1 150, 85 235

KI-385 125D4 0 230 230141C8 0 190 190339B12 0 125 125344B11 0 75 75

KI-487 352E11 0 210 210 KI-1731718C7 2 100, 40, 18 158894A9 2 100, 38, 18 156

KI-775 336A10 0 50 501209A6 0 85 85

KI-830 285F3 nd339C4 nd

KI-859 206C3 0 140 140232F7 nd237G11 0 170 170

KI-874 375G1 1 50, 45 95576G4 1 100, 50 150984G1 1 100, 90 190

KI-932 569B9 nd919A3 0 140 140

AID Genom 3994 / 6r12$$3994 02-29-96 00:53:06 gnmal AP: Genomics


TABLE 2b—Continued


KI-1017 710F4 1 110, 50 160739D3 0 75 75874E6 0 75 751016F11 nd1067C7 1 70, 50 120

F1E3 332F4 0 155 155332F4 67E10 nd

247A1 0 140 140379F8 0 85 85385C1 0 160 160415G2 0 190 190584H12 nd590E12 0 110 110655B9 nd

contigs of genomic BAC clones to anchor points on the deconvolution was achieved by dot hybridization. Forgenetic map provides an essential framework for our most of the probes, multiple BACs were identified andeffort to establish a complete, overlapping physical map arranged in putative contigs. Connectivity of BACof the human chromosomal region 22q13. New BACs clones hybridizing to one individual probe was con-identified by anonymous markers can be linked to pre- firmed by direct examination of their HindIII andexisting contigs by overlapping fragments, and outer- EcoRI restriction fingerprint patterns resolved on agar-most clones can be used to fill in gaps by chromosome ose gels for detectable overlaps and clones.walking. To extend existing contigs, insert DNA of the outer-

The first round of screening was performed on 96,000 most BAC clones of a number of contigs was collectedBAC clones arranged in a 96-well format and gridded and hybridized against the total library. To confirm theonto 42 nylon filters in a high-density 5 1 5 pattern. chromosomal localization and to rule out chimerisms,For further walking steps, an additional 75,000 clones, BACs were checked by FISH to normal metaphasereferred to as the second library (clones marked with chromosomes prior to their use as walking probes. AllB), was used. The second library is arrayed in 384 for- BACs showed a single pair of hybridization signals inmat (3072 clones per filter in 3 1 3 arrays) and extends 22q13 without any additional signals on other genomicthe entire human BAC library to approximately 71 locations (data not shown). In some of the walking stepscoverage of the sequences in the human genome. The BACs belonging to preexisting contigs were hit, indicat-initial 399 plates of the library were constructed in ing connectivity between BAC subgroups. Southern hy-pBAC108L vector (Shizuya et al., 1992), whereas the bridization with BAC DNA or the probe was performedrecombinants in the remaining plates are based on a to confirm the connectivity.modified version of this vector, pBeloBAC11, con- We identified 136 BAC clones corresponding to 19taining the lacZ gene, to enable color selection of trans- genetically mapped anchor points and 45 other mark-formants carrying insert DNA (Shizuya et al., manu-

ers located in chromosomal region 22q13. The BACsscript in preparation).were assembled into 35 contigs containing 1 to 10 indi-vidual clones with an average insert size of 145 kb

Screening the BAC Library for the 22q13 Region per BAC. A summary of the total human BAC libraryscreening results is presented in Tables 2a and 2b. Ta-For efficient library screening, 8–12 different probesble 2a shows the BAC contigs identified by geneticwere combined in one single colony hybridization, re-markers previously mapped to chromosomal regionsulting in the identification of 40–85 BACs. There were22q13 by recombination frequency analysis (Dumanskion the average 5 to 7 positive clones per probe, indicat-et al., 1991). Anchor points on the genetic map areing good agreement with an estimated 71 coverage ofdisplayed in boldface characters and can also be foundthe library used for the hybridization. Figure 1 showstogether with the BAC clones they hit on the chromo-an example of the hybridization of pooled probes tosomal map, presented in Fig. 2. Contigs identified byeight library filters. Positively identified clones wereother region-specific probes are summarized in Tablecollected and arranged in microtiter dishes to create a2b. Screening probes are shown in the left column, withchromosome 22q13-specific BAC sublibrary. The subli-corresponding BACs next to them. All grouped BACsbrary was printed onto multiple filters, and each filterform a contig. Several contigs were extended by chro-was individually hybridized to a single probe to deter-mosome walking steps, using insert DNA of outermostmine the BACs corresponding to each marker. Alterna-

tively, BAC DNA was spotted onto nylon filters, and BACs as a hybridization probe. Characterization of the


SCHMITT ET AL.16

FIG. 2. Map of chromosomal region 22q13 integrating physical coverage by BAC clones and genetic linkage information. Recombinationfraction (u) is indicated for each interval between genetic markers according to Dumanski et al. (1991). BAC contigs corresponding to thegenetic anchor points are displayed in boxes, and colocalized anonymous markers are shown next to them.

human insert DNA by PFGE after NotI digestion is mous markers D22S546/WI-424 and KI-61 (Table 2a).Hence, the three markers were placed in close genomicgiven (No. of internal NotI sites, size of NotI fragments,

and total insert size). Occasionally, when the deconvo- vicinity by sharing one BAC clone with an insert sizeof 200 kb. The markers D22S91 and D22S92 are locatedlution step was performed the same BAC clone was hit

by more than one probe, revealing a physical linkage in the chromosomal region 22q13.1 (Fig. 2). By screen-ing the BAC library, we found one clone with an insertof multiple markers, which are shown in the right col-

umn for each BAC. size of 135 kb containing both markers, indicating thatthe maximum distance between the two markers isConsidering the insert size of the smallest BAC clone

positive for certain probes, the maximum physical dis- 135 kb. We were able to place two anonymous mark-ers (D22S578/WI-17, KI-800) between D22S91 andtance of these probes on the chromosome can be pre-

dicted. For example, BAC clone 278B7 is positive for D22S92 and to extend the physical coverage towardthe telomere, thus locating D22S645/WI-382 at a dis-the genetic marker D22S84 as well as for the anony-



FIG. 3. BAC contig covering the D22S91/D22S92 area in 22q13.1. For explanations see text.

tance of approximately 100 kb distal to D22S92 (Fig. on restriction fingerprinting and Southern hybridiza-3). Overall, we were able to link nine markers with tion. Library screening with the plasmid probe D22S23/D22 Nos. (S34, S35, S326, S453, S455, S546, S578, W24F and with the two cosmids N62A6 (D22S453–S611, S645), six anonymous KI probes (KI-44, KI-61, D22Z6) and N67E5 (D22S455) revealed that BACKI-800, KI-874, KI-1210, KI-1586), and eight FISH- 115C9 is positive for all three probes. Using this BACmapped Fosmids (F1A6, F1A7, F1B11, F1E1, F1F9, as a hybridizing probe, we were able to find one over-F1G11, F2G8, F3D4) to genetically mapped markers lapping clone in each direction. A further walking step(Table 2a; Fig. 2). The anonymous probes hit BAC pulled out four more BACs, forming a contig of sevenclones that were either identified by hybridizing to ge- individual clones overall. The left panel shows the Hin-netic markers themselves or are contiguous with a BAC dIII fragment patterns of the BAC and cosmid clonescontaining a genetic anchor point. Moreover, 63 BACs belonging to this contig. Identical fragment patternswere identified by 23 additional probes, which were can be seen in several lanes representing the samebinned to chromosomal subbands using hybrid cell genomic DNA segments shared by overlapping clones.lines or FISH (Table 2b). These clones represent a Southern hybridization of BAC DNA to the HindIIIsource of regionally localized contigs, which can be used restriction patterns was performed to verify the extentto fill in gaps in case they are being hit in subsequent of the overlap between the clones (Fig. 4, center andwalking steps. Some of these probes were already right). The BACs 115C9 and 988D11 were individuallylinked to each other by hybridizing to the same BAC. hybridized to the same filter. In the center panel,

the hybridization of clone 115C9 reveals overlappingDetailed Characterization of the 22qter Region fragments in BACs 239G3 and 988D11. It also shows

that cosmids N62A6 (D22S453–D22Z6) and N67E5Figure 4 illustrates a detailed characterization of the(D22S455) are entirely embodied in 115C9. Hybridiz-most distal region of chromosome 22q. BAC contigs foring 988D11 to the same filter (right) shows the sameeach marker placed on the genetic map by linkage anal-fragments overlapping to 115C9 as in the center panel.ysis are displayed under the genomic stretch. Anony-In addition, fragment patterns shared by BAC 988D11mous markers were placed on the physical map ac-and the clones extending the contig proximally are visi-cording to hybridization data. Six BAC contigs coveringble. The same experiment was repeated with the re-about 1.5 Mb are presented. The gene encoding arylsul-striction enzyme EcoRI, and identical results werefatase A (ARSA) was mapped as the most distal markerachieved (data not shown). Combining the informationknown on the long arm of chromosome 22 (Dumanskifrom library screening with multiple probes, finger-et al., 1991), but the distance to the telomere is stillprint analysis, and Southern hybridization data, theunknown. Marker D22S163 is suspected to be locatedclones were ordered into the contig displayed in Fig. 4.approximately 120 kb distal to ARSA by PFGE and

Due to a low density of region-specific genetic mark-cosmid mapping data (H. McDermid, pers. comm.). Theers, physical mapping in the very distal area of chromo-BACs forming the ARSA contig and the BAC positivesome 22 is difficult. Chromosome walking using outer-for D22S163 (799F10) were checked for continuity bymost BACs from known contigs seems to be the onlySouthern hybridization, but no overlapping fragmentsmethod to achieve complete physical coverage ofcould be detected (data not shown). Thus, we were not22q13.3. In this study, we demonstrated the successfulyet able to fill this gap or confirm the distance anduse of several outermost BAC clones as walking probeslocalization of D22S163 distal to ARSA using the 71(Tables 2a and 2b). Gaps separating the ARSA contigBAC library.(Fig. 4) might be traced back to holes in the library,The lower panel of Fig. 4 shows the assembly of the

BAC contig surrounding genetic marker D22S23 based presumably due to unstable regions that are not clona-


SCHMITT ET AL.18

FIG. 4. Detailed physical BAC map of the telomeric region of human chromosome 22. Genetic anchor points are displayed in boldfacecharacters, and the recombination fraction (u) is indicated for each interval. (Bottom) Restriction fragment pattern and Southern hybridiza-tion of the contig surrounding marker D22S23. The HindIII digests of seven BAC clones and two cosmids (N62A6, S453 and N67E5, S455)were fractionated on 0.7% agarose gels, transferred to nylon membranes, and probed with BAC DNA. (Left) HindIII restriction fragmentpattern of BACs and cosmids. (Center) Southern hybridization of BAC 115C9 to the HindIII pattern shown to the left. (Right) Hybridizationof the same filter as that in the center with 988D11. Since miniprepped DNA of the whole BAC was used as a probe, cross-hybridizationof the vector sequences (pBAC108L, 6.7 kb; pBeloBAC11, 7.3 kb) can be seen in each lane. Signals in the cosmid lanes to the right originatefrom cross-hybridizing cos sites, as the 23.1- and 4.4-kb fragments of the l size marker also gave positive signals (not shown).

ble by partial HindIII digestion in the BAC system. clones. Failure to achieve complete physical coverageof the terminal region of chromosome 22 was also re-Another explanation could be excessive stretches of re-

peated elements that cannot be spanned by BAC ported in previously published physical maps (Cohen



artificial chromosomes with 238 mapped markers. Hum. Mol.et al., 1993; Scambler, 1994; Bell et al., 1995) exhibitingGenet. 4: 59–69.mapping problems near the long arm telomere. The

Budarf, M. L., McDermid, H. E., Sellinger, B., and Emanuel, B. S.paucity of distal markers was also noted in three librar-(1991). Isolation and regional localization of 35 unique anonymousies derived from flow-sorted chromosome 22 (Hudson DNA markers for human chromosome 22. Genomics 10: 996–1002.

et al., 1994), indicating a remarkable underrepresenta- Burke, D. T., Carle, G. F., and Olson, M. V. (1987). Cloning of largetion of this area. segments of exogenous DNA into yeast using artificial-chromosome

vectors. Science 236: 806–812.The identification of new clones that will extend con-tigs and fill in gaps by colony hybridization with BAC Chumakov, I., Rigault, P., Guillou, S., Ougen, P., Billaut, A., et al.

(1992). Continuum of overlapping clones spanning the entire hu-insert DNA is often laborious and less efficient. Newman chromosome 21q. Nature 359: 380–387.clones that share only small overlaps with the walking

Cohen, D., Chumakov, I., and Weissenbach, J. (1993). A 1st-genea-probe are easily missed, since labeling intensity oftion physical map of the human genome. Nature 366: 698–701.short fragments is often not high enough to give clear

de Jong, P. J., Yokabata, K., Chen, C., Lohman, F., Pedersen, L.,colony hybridization signals. In our experience, the McNinch, J., and Van Dilla, M. (1989). Cytogenet. Cell Genet. 51:shortest overlap detectable by colony hybridization be- 985.tween two BACs was 10%, corresponding to 15–20 kb. Delattre, O., Zucman, J., Plougastel, B., Desmaze, C., Melot, T., Pe-To create more specific hybridization probes that will ter, M., Kovar, H., Joubert, I., de Jong, P., Rouleau, G., Aurias,

A., and Thomas, G. (1992). Gene fusion with ETS DNA bindingincrease the walking efficiency, we isolated insert enddomain caused by chromosome translocation in human tumors.fragments by blunt-end ‘‘vectorette-PCR’’ using BstUI-Nature 359: 162–165.digested BAC DNA as a template (data not shown).

Driscoll, D. A., Spinner, N. B., Budarf, M. L., McDonald-McGinn,Amplified end segments of BAC inserts in a range 0.1 D. M., Zackai, E. H., Goldberg, R. B., Shprintzen, R. J., et al. (1992).to 3.0 kb are currently being used as highly efficient Deletions and microdeletions of 22q11.2 in velo-cardio-facial syn-

drome. Am. J. Med. Genet. 44: 261–268.hybridization probes to identify gap-filling BACs in ge-nomic regions deficit of genetic markers. Dumanski, J. P. (1990). Rapid procedures for the isolation of random

chromosome-specific DNA probes from a phage library. TechniqueBACs provide the faithful propagation of fairly large2: 38–42.exogenous insert fragments (ú300 kb) and can thus

Dumanski, J. P., Geurts van Kessel, A. H. M., Ruttledge, M., Wladis,be seen as intermediates between cosmids and YACs.A., Sugawa, N., Collins, V. P., and Nordenskjold, M. (1990). Isola-Their major advantages over established cloning sys- tion of anonymous, polymorphic DNA fragments from human chro-

tems are the absence of chimerisms and the easy ma- mosome 22q12–qter. Hum. Genet. 84: 219–222.nipulation of inserted DNA fragments. The successful Dumanski, J. P., Carlbom, E., Collins, V. P., Nordenskjold, M.,

Emanuel, B. S., Budarf, M. L., McDermid, H. E., Wolff, R., O’Con-screening of the BAC library with a variety of probesnell, P., White, R., Lalouel, J.-M., and Leppert, M. (1991). A maprepresenting unique genomic loci (this paper; Kim etof 22 loci on human chromosome 22. Genomics 11: 709–719.al., 1994; Neuhausen et al., 1994; Rouquier et al., 1994;

Emanuel, B., Buetow, K., Nussbaum, R., Scambler, P., Lipinski, M.,Schmitt et al., 1995) and repetitive sequences (H.and Overton, G. (1993). Report of the third international workshopSchmitt, unpublished data; Ashworth et al., 1995) to on human chromosome 22 mapping 1992. Cytogenet. Cell Genet.

assemble extended contigs proves the utility of the BAC 63: 205–211.system for the construction of detailed, long-range Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabel-

ing DNA restriction endonuclease fragments to high specific activ-physical maps of large genomes. The BAC library sup-ity. Anal. Biochem. 132: 6–13.plies an excellent source of recombinant clones for

Flint, J., Wilkie, A. O. M., Buckle, V. J., Winter, R. M., Holland,structural and functional genome characterization.A. J., and McDermid, H. E. (1995). The detection of subtelomericBACs can be used as FISH probes for cytogenetic stud-chromosomal rearrangements in idiopathic mental retardation.ies and allow the detailed molecular analysis of chro- Nature Genet. 9: 132–139.

mosomal subregions involved in mutations of medical Foote, S., Vollrath, D., Hilton, A., and Page, D. C. (1992). The humanor biological interest. Y-chromosome—Overlapping DNA clones spanning the euchro-

matic region. Science 258: 60–66.Goettert, E., Metzdorf, R., Faerber, U., and Blin, N. (1989). RegionalACKNOWLEDGMENTS

localization and molecular characterization of a DNA sequence onthe long arm of chromosome 22. Hum. Genet. 81: 385–387.We thank J. P. Dumanski (Karolinska Institute) for providing the

Gonzales, F. J., Skoda, R. C., Kimura, S., Umeno, M., Zanger, U. M.,KI probes as well as T. Hudson, S. Foote, and E. Lander (WhiteheadNebert, D. W., Gelboin, H. V., Hardwick, J. P., and Meyer, U. A.Institute/MIT Center for Genome Research) for sequence information(1988). Characterization of the common genetic defect in humanson WI STSs prior to publication. The research was funded by a grantdeficient in debrisoquine metabolism. Nature 331: 442–446.from DOE (DOE-FG03-89ER60891) to H.I.S., and H.S. was sup-

ported by a DAAD scholarship. Gough, A. C., Smith, C. A. D., Howell, S. M., Wolf, C. R., Bryant,S. P., and Spurr, N. K. (1993). Localization of the CYP2D genelocus to human chromosome 22q13.1 by polymerase chain reaction,REFERENCESin situ hybridization, and linkage analysis. Genomics 15: 430–432.

Green, E. D., Riethman, H. C., Dutchik, J. E., and Olson, M. V.Ashworth, L. K., Alegria-Hartman, M., Burgin, M., Devlin, L., Car-(1991). Detection and characterization of chimeric yeast artificialrano, A. V., and Batzer, M. A. (1995). Assembly of high-resolutionchromosome clones. Genomics 11: 658–669.bacterial artificial chromosome, P1-derived artificial chromosome,

Herman, G., Greenberg, F., and Ledbetter, D. (1988). Multiple con-and cosmid contigs. Anal. Biochem. 224: 564–571.genital anomaly/mental retardation syndrome with Goldehar com-Bell, C. J., Budarf, M. L., Nieuwenhuijsen, B. W., Barnoski, B. L.,plex due to a terminal del(22q). Am. J. Med. Genet. 29: 909–915.Buetow, K. H., et al. (1995). Integration of physical, breakpoint

and genetic maps of chromosome 22. Localization of 587 yeast Hudson, T. J., Colbert, A. M. E., Reeve, M. P., Bae, J. S., Lee, M. K.,


SCHMITT ET AL.20

Nussbaum, R. L., Budarf, M. L., Emanuel, B. S., and Foote, S. Romain, D., Goldsmith, J., Cairney, H., Columbano-Green, L.,Smythe, R., and Parfitt, R. (1990). Partial monosomy for chromo-(1994). Isolation and regional mapping of 110 chromosome 22

STSs. Genomics 24: 588–592. some 22 in a patient with del(22). J. Med. Genet. 27: 588–589.Rouleau, G. A., Haines, J. L., Bazanowski, A., Colella-Crowley, A.,Kaplan, J.-C., Aurias, A., Julier, C., Prieur, M., and Szajnert, M.-F.

Trofatter, J. A., Wexler, N. S., Conneally, P. M., and Gusella,(1987). Human chromosome 22. J. Med. Genet. 24: 65–78.J. F. (1989). A genetic linkage map of the long arm of humanKim, U.-J., Shizuya, H., de Jong, P., Birren, B., and Simon, M. I.chromosome 22. Genomics 4: 1–6.(1992). Stable propagation of cosmid sized human DNA inserts in

Rouquier, S., Batzer, M. A., and Giorgi, D. (1994). Application ofan F factor-based vector. Nucleic Acids Res. 20: 1083–1085.bacterial artificial chromosomes to the generation of contiguousKim, U.-J., Shizuya, H., Birren, B., Slepak, T., de Jong, P., and Si-physical maps: A pilot study of human ryanodine receptor genemon, M. I. (1994). Selection of chromosome 22-specific clones from(RYR1) region. Anal. Biochem. 217: 205–209.human genomic BAC library using a chromosome-specific cosmid

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). ‘‘Molecularlibrary pool. Genomics 22: 336–339.Cloning: A Laboratory Manual,’’ 2nd ed., Cold Spring Harbor Labo-

Kirschebaum, G., Chmura, M., and Rhone, D. (1988). Long arm dele- ratory, Cold Spring Harbor, NY.tion of chromosome 22. J. Med. Genet. 25: 780–783.

Scambler, P. J. (1994). Report of the fourth international workshopKouprina, N., Eldarov, M., Moyzis, R., Resnick, M., and Larionov, on human chromosome 22 mapping 1994. Cytogenet. Cell Genet.

V. (1994). A model system to assess the integrity of mammalian 67: 277–294.YACs during transformation and propagation in yeast. Genomics

Schmitt, H., Kehrer, H., Enders, H., Latos-Bielenska, A., Lamour,21: 7–17.V., Lipinski, M., and Blin, N. (1994). Involvement of 22q13.3 in

Lamour, V., Levy, N., Desmaze, C., Baud, V., Lecluse, Y., Delattre, chromosomal anomalies. Oncology Reports 1: 881–884.O., Bernheim, A., Thomas, G., Aurias, A., and Lipinski, M. (1993). Schmitt, H., Wundrack, I., Beck, S., Goett, P., Shizuya, H., Simon,Isolation of cosmids and fetal brain cDNAs from the proximal long M. I., and Blin, N. (1995). A third P-domain peptide gene (humanarm of human chromosome 22. Hum. Mol. Genet. 2: 535–540. intestinal trefoil factor) maps to 21q22.3. Submitted for publica-

Larionov, V., Kouprina, N., Nikolaishvili, N., and Resnick, M. A. tion.(1994). Recombination during transformation as a source of chime- Seizinger, B. R., Martuza, R. L., and Gusella, J. F. (1986). Loss ofric mammalian artificial chromosomes in yeast (YACs). Nucleic genes on chromosome 22 in tumorigenesis of human acoustic neu-Acids Res. 22: 4154–4162. roma. Nature 322: 644–647.

McDermid, H., Duncan, A., Brasch, C., Holden, J., Magenis, E., Shizuya, H., Birren, B., Kim, U.-J., Mancino, V., Slepak, T., Tachiri,Sheely, R., and Burn, J. (1986). Characterization of the supernu- Y., and Simon, M. (1992). Cloning and stable maintenance of 300-merary chromosome in cat eye syndrome. Science 232: 646–648. kilobase-pair fragments of human DNA in Escherichia coli using

Narahara, K., Takahashi, Y., Murakami, M., Tsuji, K., Yokoyama, F-factor-based vector. Proc. Natl. Acad. Sci. USA 89: 8794–8797.Y., Murakami, R., Ninomiya, S., and Seino, Y. (1992). Terminal Stein, C., Gieselmann, V., Kreysing, J., and von Figura, K. (1989).22q deletion associated with a partial deficiency of arylsulfatase Cloning and expression of human arylsulfatase A. J. Biol. Chem.A. J. Med. Genet. 29: 432–433. 264: 1252–1259.

Nesslinger, N., Gorski, J., Kurczynski, T., Shapira, S., Siegel-Bartelt, Sternberg, N. (1990). Bacteriophage P1 cloning system for the isola-J., Dumanski, J., Cullen, R., French, B., and McDermid, H. (1994). tion, amplification, and recovery of DNA fragments as large as 100Clinical, cytogenetic and molecular characterization of seven pa- kilobase pairs. Proc. Natl. Acad. Sci. USA 87: 103–107.tients with deletions of chromosome 22q13.3. Am. J. Hum. Genet. Trofatter, J. A., Rouleau, G. A., Gusella, J. F., and Haines, J. L.54: 464–472. (1990). Extended genetic linkage map of human chromosome 22.

Neuhausen, S. L., Swensen, J., Miki, Y., Liu, Q., Tavtigian, S., Shat- Am. J. Hum. Genet. 47: A201.tuck-Eidens, D., Kamb, A., Hobbs, M. R., Gingrich, J., Shizuya, Watt, J., Olson, I., Johnston, A., Ross, H., Couzin, D., and Stephen,H., Kim, U.-J., Cochran, C., Futreal, P. A., Wiseman, R. W., Lynch, G. (1985). A familial pericentric inversion of chromosome 22 withH. T., Tonin, P., Narod, S., Cannon-Albright, L., Skolnick, M. H., a recombinant subject illustrating a pure partial monosomy syn-and Goldgar, D. E. (1994). A P1-based physical map of the region drome. J. Med. Genet. 22: 283–287.from D17S776 to D17S78 containing the breast cancer susceptibil- Wilson, D. I., Burn, J., Scambler, P., and Goodship, J. (1993). Syn-ity gene BRCA1. Hum. Mol. Genet. 3: 1919–1926. dromes of the month: DiGeorge syndrome, part of CATCH22. J.

Nizetic, D., Gellen, L., Hamvas, R. M. J., Mott, R., Grogoriev, A., Med. Genet. 30: 852–856.Vatcheva, R., Zehetner, G., Yaspo, M.-L., Dutriaux, A., Lopes, C., Xie, Y.-G., Han, F.-Y., Bajalica, S., Blennow, E., Kristoffersson, U.,Delabar, J.-M., Van Broeckhoven, C., Potier, M.-C., and Lehrach, Dumanski, J. P., and Nordenskjoeld, M. (1994). Identification,H. (1994). An integrated YAC-overlap and ‘cosmid-pocket’ map of characterization and clinical applications of cosmids from the te-the human chromosome 21. Hum. Mol. Genet. 3: 759–770. lomeric and centromeric regions of the long arm of chromosome

Phelan, M. C., Thomas, G. R., Saul, R. A., Rogers, C., Taylor, H. A., 22. Hum. Genet. 94: 339–345.Wenger, D. A., and McDermid, H. E. (1992). Cytogenetic, biochemi- Yubisui, T., Naitoh, Y., Zenno, S., Tamura, M., Takeshita, M., andcal, and molecular analyses of a 22q13 deletion. Am. J. Med. Genet. Sakaki, Y. (1987). Molecular cloning of cDNAs of human liver and43: 872–876. placenta NADH-cytochrome b5 reductase. Proc. Natl. Acad. Sci.

USA 84: 3609–3613.Pierce, J. C., Sauer, B., and Sternberg, N. (1992). A positive selectionvector for cloning high molecular weight DNA by the bacteriophage Zheng, L. M., Andrews, P. C., Hermodson, M. A., Dixon, J. E., andP1 system: Improved cloning efficacy. Proc. Natl. Acad. Sci. USA Zalkin, H. (1990). Cloning and structural characterization of por-89: 2056–2060. cine heart aconitase. J. Biol. Chem. 265: 2814–2821.

Zucman, J., Delattre, O., Desmaze, C., Epstein, A., Stenman, G.,Riond, J., Mattei, M. G., Kaghad, M., Dumont, X., Guillemot, J. C.,le Fur, G., Caput, D., and Ferrara, P. (1991). Molecular cloning Speleman, F., Fletchers, C., Aurias, A., and Thomas, G. (1993).

ETS and ATF1 gene fusion induced by t(12;22) translocation inand chromosomal localization of a human peripheral-type benzodi-azepine receptor. Eur. J. Biochem. 195: 305–311. malignant melanoma of soft parts. Nature Genet. 4: 341–344.


Framework for a Physical Map of the Human 22q13 Region Using Bacterial Artificial Chromosomes (BACs)

Documents

Transcript of Framework for a Physical Map of the Human 22q13 Region Using Bacterial Artificial Chromosomes (BACs)